Microneedles loaded with glutathione‐scavenging composites for nitric oxide enhanced photodynamic therapy of melanoma

Abstract Photodynamic therapy (PDT) represents an attractive promising route for melanoma treatment. However, its therapeutic efficacy is compromised by inefficient drug delivery and high glutathione (GSH) levels in cancer cells. To overcome these challenges, microneedles (MNs) system loaded with GSH‐scavenging nanocomposites was presented for nitric oxide (NO) enhanced PDT. The nanocomposites consisted of S‐nitroso‐N‐acrylate penicillamine (SNAP; a NO donor) grafted fourth‐generation polyamide amine dendrimer (G4) and chlorin e6 (Ce6). Upon local insertion of polyvinylpyrrolidone MNs, G4‐SNAP/Ce6 composites were fast delivered and significantly amplified the therapeutic effects during PDT, via GSH depletion and reactive nitrogen species generation. Even with a single administration and low power light exposure, MNs with G4‐SNAP/Ce6 effectively halt the tumor progression. The system demonstrated better cancer ablation efficacy than Ce6 alone toward melanoma. The strategy may inspire new ideas for future PDT‐related therapy for skin tumors.


| INTRODUCTION
Melanoma is one of the most aggressive cancers in humans, especially in aging people. The rapid progression contributes to its malignancy, causing a rather unsatisfied 5-year survival rate in clinics. [1][2][3] Many treatments are developed to fight against melanoma cancer. Among them, photodynamic therapy (PDT) has attracted rising attention both in research and clinical applications. [4][5][6][7] During standard PDT, photosensitizers (PS) are intravenously administrated and subsequently activated by light in tumor sites to produce tumor-destroying reactive oxygen species (ROS). [8][9][10] However, systemic administration of PS leads to limited drug enrichment in targeted lesions of tumors. 11 It unavoidably brings about an accumulation of PS in healthy superficial organs or tissues as a side effect. 12,13 When exposed to daylight, the generated ROS damages the DNA of normal skin cells and ironically promotes the potential occurrence of skin cancer. Besides, tumor cells produce a high level of intracellular glutathione (GSH). [14][15][16] As a naturally occurring reductant, GSH is a potent scavenger of various oxidants. It protects cancer cells from ablation of ROS, thus dampening the outcome of PDT. There is always a long-lasting need to exploit new material strategies with higher delivering precision and GSHscavenging ability to enhance PDT.
Microneedles (MNs) have emerged as an innovative platform for efficient transdermal drug delivery in recent years. [17][18][19] They are quite flexible in cargo selection, facile in manufacture, and minimally invasive to the patients. [20][21][22][23][24][25] More importantly, local administration gives MNs a peerless edge in the accuracy of drug delivery, making it an ideal method to treat melanoma tumors and other cutaneous illnesses. [26][27][28][29][30] It is an excellent choice to realize targeted delivery of PS and to avoid the side effects of PDT. Fan Jia and Weijiang Yu contributed equally to this study.
On the other hand, S-nitrosothiols (RSNOs) are a large family of compounds, many of which are found in the human body as native carriers for nitric oxide (NO) over a long distance. [31][32][33] They are generally considered to have good safety and can release NO by consuming a high level of GSH inside cells. [34][35][36][37] Interestingly, NO can reduce intracellular GSH levels via various bio-metabolism as well. [38][39][40] It can also react with ROS to produce peroxynitrite anions (ONOO À ) or other reactive nitrogen species (RNS), which are more lethal than either ROS or NO. 41,42 Such benefits make RSNOs a promising choice for PDT to resist consumption by GSH and boost efficiency.
Hence, in the present study, we combined NO gas therapy and MNs to overcome the drawbacks of PDT mentioned above. GSH responsive NO donor, S-nitroso-N-acrylate penicillamine (SNAP), was conjugated onto the outer amine groups of fourth-generation polyamidoamine dendrimer (G 4 -NH 2 ) to yield a GSH consuming scaffold. The positive charges of the SNAP modified dendrimer could attract and pack negatively charged chlorine 6 (Ce6) inside via gentle procedure, without compromising the attached NO. Since NO is highly susceptible to scavenging, such a coloading method is beneficial to maximizing synergistic effects between NO and PDT. The Ce6/NO co-loaded dendrimer was then encapsulated in dissolving MNs, using biocompatible polyvinylpyrrolidone (PVP) as the matrix. We hypothesized that the SNAP and subsequently produced NO could deplete the high-level GSH inside melanoma tumor cells upon topical inoculation of MNs, thus improving the therapeutic outcome of the following PDT (Scheme 1). The present work might provide new inspiration for future skin cancer therapy.   1 H NMR spectra were obtained from an NMR spectrometer (DMX 500; Bruker). Morphology, particle size distribution, and zeta potential of nanoparticles were obtained on transmission electron microscopy (HT7700; Hitachi) and Zetasizer Nano-ZS (Malvern). UVvis spectra were recorded on a UV-vis spectrometer (UV-2550; Shimadzu).

| Cell culture
Human melanoma A375 cells were purchased from Tong Pai

| GSH measurement
Cellular GSH levels were determined by the mercapto assay kit (BC1370; Solarbio). The thiol groups on GSH could react with 5,5 0dithiobis-(2-nitrobenzoic acid) and generate a yellow compound that had maximum absorption at 412 nm. To study the GSH depletion ability of G 4 -SNAP/Ce6, different volumes of G 4 -SNAP/Ce6 solutions were directly added into GSH solutions with final NO/GSH molar concentration ratios at 0, 0.2, 0.5, and 1, respectively. To study the intracellular GSH levels, A375 cells were seeded into 24-well plates at a density of 10 5 cells per well. After incubating with G 4 /Ce6 (Ce6 10 μg/ml), G 4 -SNAP (NO 10 μg/ml), G 4 -SNAP/Ce6 (Ce6 10 μg/ml and NO 10 μg/ml) for 3 h, the cells were washed with PBS and lysed by repeated freeze-thaw. The GSH levels of samples were measured by the absorbance at 412 nm using a UV-vis spectrometer (UV-2550; Shimadzu).
For tumors, excised tissues were quenched in liquid nitrogen and subjected to freeze-drying. 10 mg of dry tissues were homogenated into suspension in 100-μl PBS and centrifuged. The supernate was collected and protein levels were determined to standardize the number of samples before the test. The GSH level was measured similarly to the mentioned above. For total GSH concentration, samples were first incubated with NADPH, before being measured.

| GSH triggered NO release
To investigate GSH triggered NO release from G 4 -SNAP/Ce6, 20 μl of PBS with different GSH concentrations were added to 1.98 ml G 4 -SNAP/Ce6 solution (NO 100 μg/ml), with a final GSH concentration of 0 μM, 2 μM, and 5 mM, respectively. The NO release profiles were recorded by Free Radical Analyzer (TBR 4100; WPI) equipped with a NO detector.  For tumors, 50-mg excised tissues were homogenated into suspension in 500 μl PBS and centrifuged. The supernates were collected and protein levels were determined to standardize the number of samples before the test. ROS levels were determined by an O12 fluorescent probe (maximum λ ab = 488 nm, λ em = 526 nm) on a SpectraMax M5/M5e microplate system (Molecμlar Devices). RNS was measured similarly with O52 as a probe (maximum λ ab = 488 nm, λ em = 530 nm).

| Cytotoxicity assay
The cell cytotoxicity on A375 cells was performed by CCK-8 assay.
A375 cells were seeded into 96-well plates at a density of 8000 cells in 200 μl DMEM medium per well. After 24 h incubation, cells were treated with different concentrations of Ce6, G 4 -SNAP, and G 4 -SNAP/ Ce6, respectively, and incubated for another 3 h. After replacing with a fresh medium, cells were treated either with or without 660 nm light (0.2 W/cm 2 , 1 min) radiation and incubated for another 24 h. Then, CCK-8 (20 μl/well) was added for another 4-h incubation. Finally, OD450 was measured by a microplate reader (Thermo Fisher Scientific).  These slices were stained with hematoxylin and eosin (H&E) staining for histological analysis. To observe the drug diffusion, the slices were observed by a fluorescence microscope (DS-Ri2; Nikon).

| Animals and tumor model
Male Balb/c nude mice (4-week-old) were purchased from the Zhe-

| Statistics
All experiments were conducted with at least three replicates. All experimental data were presented as their mean ± SD and analyzed by one-way ANOVA. *p < 0.05 was considered statistically significant.

| Synthesis and fabrication of SNAP/Ce6 coloaded dendrimer
Different RSNOs have various stability. Their NO release half-lives may range from a few seconds to tens of hours. 43 The more stable the RSNOs are, the fewer NO losses before administration, hence the higher the delivering efficacy is. SNAP is one of the most stable RSNOs, due to unique molecular structures on its γ-C. 44,45 The SNAP dendrimer (G 4 -SNAP) was obtained via a three-step process (Figures 1a and S1). The successful synthesis of each step was confirmed by 1 H NMR results (Figures 1b and S2). The average grafting number on each dendrimer was calculated as 55 (conjugating ratio 86%). It was based on the ratio between the accumulated intensity of peaks at δ = 3.20 ppm and 1.25 ppm (Figure 1b), which belonged to methylenes of G 4 dendrimer and methyl groups of SNAP, respectively.
The zeta potential decreased from 30 to 4 mV after encapsulation of negatively charged Ce6 in the dendrimer (Figure 1c). UV-Vis spectrometry was further employed to monitor the formation of the G 4 -SNAP/Ce6 composites. The peak at 343 nm in spectra corresponds to the characteristic absorbance of SNAP, and the peaks at 500, 660, and 400 nm correspond to the characteristic absorbance of Ce6 (as labeled in Figure 1d). The mass ratio of Ce6 in the G 4 -SNAP/Ce6 composites was measured by UV-Vis spectrum at 660 nm and calculated as 6% according to a standard curve of Ce6 ( Figure S3). The average hydrodynamic diameter of dendrimer increased from 13 to 21 nm, as indicated by DLS (Figure 1e). All these data suggested the successful loading of Ce6 in G 4 -SNAP.

| GSH initiated NO release and intracellular delivery of Ce6
It is reported that the GSH concentration inside cells is thousands of times higher than that outside cells (5-10 mM vs. 2 μM). Such a huge difference is ideal as a trigger for specific NO release (Figure 2a). The

| Depletion of GSH by G 4 -SNAP/Ce6
The GSH consumption by G 4 -SNAP/Ce6 was critical in improving the efficiency of subsequent PDT. As determined by the Ellman assay using a standard curve method ( Figure S5), the remaining GSH in PBS decreased constantly with the elevated ratio between RSNOs and GSH. When the molar concentrations of RSNOs and GSH were equal, about 90% of GSH was eliminated only after 30 min incubation (Figure 2d). The GSH concentration in cells also reduced dramatically to less than 50% after being cultured with either G 4 -SNAP or G 4 -SNAP/Ce6 kept in the dark for 3 h (Figure 2e). However, the GSH depleting ability was entirely lost, when G 4 -SNAP/Ce6 was heated to 60 C for 4 h before cellular incubation to prematurely disrupt RSNOs and release NO (92%; Figure S6). The results demonstrated the necessity of G 4 -SNAP in depleting GSH. It also proved G 4 -SNAP as a favorable GSH scavenger.

| Cell toxicity evaluation
The cell viability was then quantified using a standard CCK-8 assay.
Without Furthermore, the safety of the therapy was evaluated by a complete blood panel test and histological analyses of major organs and skin tissue (MN-treated site). The blood test did not demonstrate noticeable changes in cell counts of red blood cells, white blood cells, blood platelet, lymphocyte, monocyte, and neutrophil, respectively ( Figure S8). The results indicated that the therapy had desirable blood compatibility, nor did it promote significant immunological responses. Meanwhile, histological staining slices of major organs and skin tissues also presented negligible damages after different treatments ( Figure S9). All these data proved the good safety of PDT mediated by G 4 -SNAP/Ce6 MNs.

| In vivo detection of GSH, RNS, and apoptotic markers
The GSH concentration of tumors with different treatments was further quantified. As shown in Figure